DESCRIPTION: The PI, Dr. Dirk de Rooij, requests three years of support to investigate factors influencing spermatogonial stem cell growth and differentiation in culture. Spermatogonial stem cells will be purified from testes of vitamin A-deficient (VAD) mice. The first aim is to develop a culture system for maintaining long-term viability of these highly purified and undifferentiated spermatogonia. The second aim is to develop culture conditions that will promote proliferation of spermatogonia. The third aim is to establish immortalized cell lines of undifferentiated cells, using transfection with immortalizing genes if necessary. The fourth aim is to find the factor(s) that will induce differentiation of the cultured spermatogonia. The final aim is to test the developmental competence of these cells by studying the effects of various intervals and conditions of culture on the ability of the spermatogonial stem cells (and stem cell lines) to repopulate germ cell-deficient testes after transplantation.